In mammalian heart MgATP stimulates the Na⁺/Ca²⁺ exchanger (NCX1) by increasing the apparent affinity for cytosolic Ca²⁺. This take place only in the presence of vanadate, but if the phosphorylating analogue ATP-γ-S is used vanadate is not longer required. The non-phosphorylating analogue AMP-PCP is totally ineffective. In no case the carrier becomes phosphorylated. This nucleotide stimulation is not affected by unspecific protein alkaline phosphatase but is abolished by a phosphatidylinositol specific phospholipase C (PtdIns-PLC). PtdIns counteracts PtdIns-PLC inhibition. In the absence of ATP, PtdIns-4,5-P₂ and in less extent PtdIns-4-P, stimulate NCX1. The  process  related  to  ATP  stimulation involves  a  fast  phosphorylation  chain: a Ca²⁺ independent  PtdIns-4-P formation and followed by a Ca²⁺ dependent synthesis of PtdIns-4,5-P². Western blots and cross immunoprecipitation analysis demonstrates that PtdIns-4,5-P₂ binds strongly to NCX1. ATP, Ca²⁺, vanadate and PtdIns-PLC modulate this binding in a way resembling the ATP regulation of the exchange fluxes. Taken together, this indicates that in bovine heart sarcolemmal  vesicles, ATP up regulation of NCX1 is related to  increase of the amount of PtdIns-4,5-P₂ bound to the exchanger.